Immunobiology 223 (2018) 477–485

Contents lists available at ScienceDirect

Immunobiology
journal homepage: www.elsevier.com/locate/imbio

Review

Applications of chemokines as adjuvants for vaccine immunotherapy T

⁎
Teena Mohan, Wandi Zhu, Ye Wang, Bao-Zhong Wang
Center for Inflammation, Immunity and Infection, Institute for Biomedical Sciences, Georgia State University, 100 Piedmont Ave SE, Atlanta, GA 30303, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Vaccinations are expected to aid in building immunity against pathogens. This objective often requires the
Adjuvants addition of an adjuvant with certain vaccine formulations containing weakly immunogenic antigens. Adjuvants
Chemokines can improve antigen processing, presentation, and recognition, thereby improving the immunogenicity of a
Cytokines vaccine by simulating and eliciting an immune response. Chemokines are a group of small chemoattractant
Vaccination
proteins that are essential regulators of the immune system. They are involved in almost every aspect of tu-
Immune responses
morigenesis, antitumor immunity, and antimicrobial activity and also play a critical role in regulating innate and
adaptive immune responses. More recently, chemokines have been used as vaccine adjuvants due to their ability
to modulate lymphocyte development, priming and effector functions, and enhance protective immunity.
Chemokines that are produced naturally by the body’s own immune system could serve as potentially safer and
more reliable adjuvant options versus synthetic adjuvants. This review will primarily focus on chemokines and
their immunomodulatory activities against various infectious diseases and cancers.

1. Introduction responses against co-administered antigens. Adjuvants increase im-

mune responses to vaccines, enhance protection against pathogens,
The goal of vaccination is the generation of strong and long-term increase the speed of primary immune responses, activate the appro-
protection to a specific pathogen. This is accomplished by eliciting ro- priate type of immunity to a given threat, increase the generation of
bust immune responses to a targeted antigen which can induce long- memory responses, and can alter the specificity and breadth of the
term protection against infection. This target antigen could be a la- generated immune responses (Vogel, 1998). Adjuvants can also im-
boratory generated or altered pathogen, virus, bacteria, protein, or prove immune responses in populations where responses to vaccines
other foreign substance. These antigens can trigger responses from the are typically reduced, such as infants, the elderly, and im-
host immune system that result in long-term immunity in the process. munocompromised patients. The functional outcomes of enhancing
Certain vaccines, often containing live-attenuated antigens, are effec- immune responses are manifold, including the allowance for reductions
tive because they can stimulate the innate immunity to cascade into in the quantity of antigen contained in individual vaccine dose. This
subsequent adaptive immune responses capable of clearing the targeted combination of less antigen and fewer doses can improve the vaccine
pathogens (Hoebe et al., 2004). New developments in the field of supply worldwide and reduce the total cost of vaccine production.
vaccinology include DNA vaccines, novel peptides, recombinant viruses There are several adjuvants that are currently in the market or in
and proteins, and conjugates, all of which have been introduced com- development but aluminum based adjuvants including aluminum hy-
mercially. This new generation of vaccines is safer and has fewer ne- droxide, aluminum phosphate, and alum still lead the way. Though
gative reactions or side effects than previous generations of vaccines alum has been known to cause type-1 hypersensitivity reactions post-
made from live or whole-inactivated organisms. Conversely, these new administration in a small percentage of patients, it is among the few
advancements are sometimes not effective when a pathogen’s natural adjuvants that have been approved by the Food and Drug
infection pathway does not convey long-lasting immunity (Hilleman, Administration (FDA) for human use (Gupta et al., 1995). Aluminum
2000). To achieve the desired goal of protective immunity in these salts that have met FDA approval are aluminum hydroxide, aluminum
cases, the addition of a unique adjuvant to the vaccine is needed. phosphate, potassium aluminate sulfate, and mixed aluminum com-
pounds. A significant number of other identified adjuvants are clearly
2. Adjuvants more potent than alum but toxicity is the single most important im-
pediment in introducing most such adjuvants to human use (Edelman,
Adjuvants are molecules or compounds that can enhance immune 1980). Some licensed vaccines containing alum or other adjuvants

⁎
Corresponding author.
E-mail address: [email protected] (B.-Z. Wang).

https://doi.org/10.1016/j.imbio.2017.12.001
Received 3 November 2017; Received in revised form 7 December 2017; Accepted 7 December 2017
Available online 08 December 2017
0171-2985/ © 2017 Elsevier GmbH. All rights reserved.
T. Mohan et al. Immunobiology 223 (2018) 477–485

include: ASO4, comprised of aluminum hydroxide and monophosphoryl vertebrate animals as well as in some microbes including bacteria and
lipid A (MPL), is used to treat cervical cancer caused by human pa- viruses, but none have yet been identified in other non-vertebrates
pillomavirus (HPV) (Tagliabue and Rappuoli, 2008); AS03, comprised (Wiedle et al., 2001). Historically, chemokines have been known by
of oil compounds, vitamin E and squalene, is used in an influenza various
vaccine against H5N1 (Leroux-Roels, 2009). In Europe, MF59 is an names including the SIS family, SIG family, SCY family, the inter-
adjuvant component of influenza vaccine for elderly patients (Fluad, crines, and the PF4 superfamily. The action of a chemokine is mediated
Novartis Vaccines) (Mulligan et al., 2014) and AS04 (combination of through chemokine receptors, members of the G protein-coupled re-
alum and MPL, GlaxoSmithKline) is the adjuvant for some viral vac- ceptor (GPCR) family, which can stimulate signal transduction path-
cines, e.g. Hepatitis B virus (HBV) and HPV (Monie et al., 2008). ways inside the cell when they are activated (Zhang and LiWang, 2014).
To date, there are more than 50 chemokines and 18 chemokine re-
3. Downsides of synthetic adjuvants ceptors identified. These molecules are classified into four families (CC,
CXC, C, and CX3C) based on the way the first two conserved cysteine
Aluminum based synthetic adjuvants continue to be the dominant residues are arranged, creating a structural motif (Bachelerie et al.,
adjuvants used. However, these synthetic adjuvants can sometimes 2014a; Bachelerie et al., 2014b). Chemokines have evolved to become
trigger unwanted “hyperactive” immune responses, causing the im- an integral part in the many critical roles involved in regulating innate
mune system to over-react to antigens found in the vaccine formula- and adaptive immune responses (Mariani and Panina-Bordignon,
tions. This increases the probability of disrupting the host’s self-toler- 2003). Chemokines modulate lymphocyte development, priming and
ance, leading the immune system to attack its own cells and tissues. It effector functions, and play a vital role in immune surveillance. Many
has been established that vaccine induced inflammatory responses can chemokines have been shown to be effective immunological adjuvants
lead to permanent alterations in immune functionality and increase the in a variety of model systems, enhancing protection induced by viral,
risk of developing immunological disorders, including autoimmunity bacterial, and parasitic vaccines (Smit and Lukacs, 2006). These che-
and long-term inflammation. Additionally, despite almost 80 years of mokines have also increased various immunological parameters in
widespread use, our knowledge behind the mechanisms of aluminum tumor immunization models and clinical trials (Esche et al., 2005).
adjuvants is still remarkably limited. An equally concerning lack of data Chemokines types, binding to their respective receptors and their
on the pharmacokinetics and toxicology of these compounds also re- generalized functions have been demonstrated in Fig. 1.
mains problematic. To date, certain new and innovative adjuvants have
shown acceptable safety profiles in clinical trials across a variety of 5. Immunomodulatory studies of chemokines
applications and in post-licensure experiences, however many of these
adjuvants still possess disadvantages such as high-costs, lack of stability More recently, chemokines have found a use as vaccine adjuvants,
and reliability, and most importantly high-toxicity (Tomljenovic and due to their ability to regulate immune responses and enhance the
Shaw, 2011; Aomljenovic, 2011). protective immunity (Eo et al., 2001; Lu et al., 1999; Sin et al., 2000).
Chemokines/cytokines, which are produced naturally by the body’s Chemokine adjuvants can modulate the direction and magnitude of
own immune system, could serve as potentially safer and more reliable induced immune responses generated by DNA, protein, subunit or
adjuvant options versus synthetic adjuvants. Vaccine safety, effective- peptide vaccines. However, most of the chemokine adjuvants are cur-
ness, and toxicity could all be improved by using immunostimulators rently struggling with dose-related toxicity issues. Additionally, re-
that are naturally non-toxic, have well understood mechanisms, and are combinant chemokines have displayed stability problems, giving them
adaptable to many types and modes of vaccines. The current review is short half-lives and limiting their usefulness as vaccine adjuvants. Some
concerned with the importance of natural and non-toxic adjuvants and of these limitations can be overcome by utilizing liposome or micro/
their widespread use in the vaccine industry. Published studies on the nanoparticle encapsulation and co-administering chemokine expression
relevant topics were analyzed and compared to create a composite view vectors with DNA or protein-based vaccines, respectively.
of the topic. In this review, we have emphasized the role of various With an array of chemokines and ligands to select from to target on
chemokines as adjuvants in the treatment of many diseases and cancers. immune cells, it becomes possible to tailor a vaccine’s effects by che-
moattracting specific cell types. For example, antigen presenting cell
4. Cytokines and chemokines (APCs) targeting approaches have been evaluated for influenza and HIV
antigens. In these studies, antigenic delivery with chemokines such as
Cytokines are a broad category of small proteins (∼5–20 kDa) that CCL3 have resulted in enhanced immunogenicity and protection against
are important in cell signaling (Boyaka and McGhee, 2001). Cytokines these pathogens. The chemokine CCL3 has demonstrated the ability to
include chemokines, interferons, interleukins, tumor necrosis factor, attract natural killer (NK) and CD8+ T cells, and has been investigated
and lymphokines. Traditional adjuvants’ mode of action is to stimulate as an adjuvant for effective vaccines (Allen et al., 2017).
the creation of an environment that promotes immunity. For example, In the CCL3 study, researchers expressed CCL3 together with a
aluminum salts and Freund’s adjuvants have been linked to IFN-γ, IL-2, truncated HIV-1 Gag antigen in L. plantarum and observed a significant
or IL-12 for establishing both innate and adaptive immunity. Instead of increased recruitment of T cells. Another finding strongly supports that
using an adjuvant to induce the production of specific cytokines, these the co-delivery of CCL3 by an adenovirus-based vaccine improves
cytokines can now be used directly as adjuvants either alone or in protection from retrovirus infection (Kuczkowska et al., 2015). In the
combination with other adjuvants (Grob et al., 1984; Kim et al., 2001). Friend retrovirus (FV) mouse model, CCL3 was co-expressed from
In one of our recent findings, we also studied a novel cytokine, adenoviral vectors, together with FV Gag and Env antigens. Env and
GIFT4, engineered by fusing GM-CSF and IL-4. We observed that GPI- Gag-Pol antigens coadministered with a vector encoding CCL3 induced
anchored GIFT4 containing virus-like particles (VLPs) induced higher higher virus-specific antibody titers and CD4+ T cell responses
levels of systemic antibody responses with significantly increased (Kuczkowska et al., 2015).
binding avidity and improved neutralizing breadth and potency to a In addition, vaccine formulations targeting XCR1 on cross-pre-
panel of selected strains, as well as higher levels of IgG and IgA at senting dendritic cells (DCs) using antigen fused to XCL1, the only
several mucosal sites (Feng et al., 2015). known ligand for XCR1, induce protective CD8+ T cell responses
Chemokines are a family of chemoattractant cytokines which play a against influenza virus and strengthen the role of chemokines in mod-
vital role in cell migration and in induction of cell movement in re- ulating host immunity (Fossum et al., 2015).
sponse to a chemical gradient by a process known as chemotaxis. Recently, we investigated the effects of GPI-anchored CCL28, a
Chemokines are small proteins (8–10 kDa) and are present in all chemokine associated with mucosal surfaces, as an adjuvant with

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Fig. 1. Chemokines at a glance: Figure represent the (A) types of various chemokines; (B) Binding of chemokines with their receptors; and (C) functions of various chemokines.

influenza VLPs in mice. We observed that the membrane bound form of in the ‘Homing of B and T cell’ section.
CCL28 in influenza VLPs acted as a strong immunostimulator at both
systemic and mucosal sites, and boosted a significant cross-protection in 6. Chemokines and cancer vaccines
animals against heterologous viruses across a large distance (Mohan
et al., 2017; Mohan et al., 2016). It was demonstrated that the use of Current cancer vaccine approaches have failed to reliably induce
CCL28 as an adjuvant has unique immunity modulating properties at robust B and T cell activation. It is thought that a lack of sufficient
various mucosal compartments. CCL28 attracts IgA, but not IgG or IgM physical interaction with the necessary immune cells could be the
producing cells, and promotes their migration to different mucosal sites cause. Chemokines, can increase the recruitment of relevant immune
(Lazarus et al., 2003). Because of its specific role in orchestrating the cells, e.g. DCs, macrophages, lymphocytes, and NK cells, directed to-
localization of IgA antibody secreting cells (ASCs), we also analyzed the ward tumor cells through their chemotactic properties. Over the last
long-lasting mucosal adjuvanticity of GPI-anchored CCL28 co-in- few decades, chemokines have been found to be involved in almost
corporated in influenza VLPs. Our results suggest that the CCL28 in- every aspect of tumorigenesis, antitumor immunity, and antimicrobial
duces significantly higher mucosal antibody responses involved in activity.
providing long-term cross-protection against H3N2 influenza viruses In a cancer immunity study, CCL3- and CCL20-recruited DCs were
when compared to other vaccination groups (Mohan et al., 2017). modified by adenovirus-transduced tumor-associated antigen, MAGE-1,
In a separate report, rhesus macaques vaccinated with SIV DNA and and shown to stimulate anti-tumor immunity specific to gastric cancer
CCR9L (CCL25) or CCR10L (CCL28/CCL27) adjuvants showed sig- ex vivo and in vivo. This system demonstrated the involvement of che-
nificant protection from multiple low-dose intravaginal challenges with mokines in recruiting DCs and may prove to be an efficient strategy for
SIVsmE660. In this study, four groups of animals consisting of five fe- anti-tumor immunotherapy (He et al., 2010).
male rhesus macaques with pSIVmac239 pol and pSIV sooty mangabey In another report, mice with a glioma tumor were given DCs pulsed
were vaccinated with consensus Env and gag vaccine alone or in with glioma cell lysate subcutaneously, while simultaneously being
combination with CCR9L pCCL25 or CCR10Ls pCCL28 or pCCL27. given an intratumoral injection of a plasmid coding for CXCL10. The
Sixty-eight percent of all vaccinated animals remained either unin- mice given the combination therapy containing CXCL10 displayed
fected or had aborted infection compared to only 14% in the vaccine significantly increased survival rates. The increased survival or potent
naïve group. The highest protection was observed in the CCR10L che- antitumor immune responses could be directly correlated with che-
mokines group, where 6 of 9 animals had aborted infection and two motactic properties of CXCL10 towards activated dendritic and T cells
remained uninfected, leading to 89% protection. The induction of (Li et al., 2007).
mucosal SIV-specific antibodies and neutralization titers correlated CCL21, which enhances Th1 responses and assists in T cell migra-
with trends in protection and these results indicate the role of chemo- tion to secondary lymph organs has been another popular cancer vac-
kine in modulating immune responses (Kutzler et al., 2016). cine adjuvant. Mice who were injected with CCL21 into a hepatocel-
Involvement of many other chemokines in the attraction of various lular carcinoma displayed improved survival, slowed tumor
immune cells were also discussed in detail later in the review especially progression, and increased intratumoral T cell penetration (Nguyen-

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Table 1
Role of Chemokines as adjuvants/immunostimulators in different diseases.

Chemokines Other name(s) Receptor Pathogen/Diseases References

CCL1 I-309 CCR8 HIV/SIV. Tsuji et al. (1997)

TCA-3
CCL2 MCP-1 CCR2 Arthritis, Autoimmune diseases, neurodegenerative disorders such as Bose and Cho (2013), Sin et al. (2000), Eo et al. (2001),
Alzheimer's disease, Multiple sclerosis, Ischemic brain injury, HSV, Nagai and Masuzawa (2001)
Glioma.
CCL3 MIP-1α CCR1 HSV, HIV/SIV, Gastric cancer, Influenza, Plasmacytoma, Multiple Sin et al. (2000), Eo et al. (2001), Lu et al. (1999), He
myeloma, B cell lymphoma. et al. (2010), Fredriksen and Bogen (2007), Ruffini et al.
(2010)
CCL4 MIP-1β CCR1 HSV. Eo et al. (2001)
CCR5
CCL5 RANTES CCR5 HSV, Lymphoma, Myelomonocytic leukemia. Sin et al. (2000), Kutubuddin et al. (1999), Inoue et al.
(2008)
CCL7 MARC CCR2 Melanoma, Human Wetzel et al. (2007), Wetzel et al. (2001), Hu et al.
MCP-3 cervical carcinoma, Colorectal cancer, Mycobacterium tuberculosis, (2002), Ryan et al. (2007), Fioretti et al. (1998)
Mastocytoma.
CCL16 LEC CCR1 Mammary carcinomas. Giovarelli et al. (2000)
NCC-4 CCR2
LMC CCR5
Ckβ12 CCR8
CCL17 TARC CCR4 Melanoma, Colon carcinoma. Okada et al. (2006), Kanagawa et al. (2007)
Dendrokine ABCD-
2
CCL19 ELC CCR7 HCV, HBV, Fibrosarcoma, Adenocarcinoma, Breast cancer, Ovarian Hartoonian et al. (2014), Westermann et al. (2007), Hou
carcinoma. et al. (2009), Braun et al. (2000), Gao et al. (2005)
Exodus-3 Ckβ11
CCL20 LARC CCR6 Gastric cancer. He et al. (2010)
Exodus-1 Ckβ4
CCL21 SLC CCR7 Colon cancer, Melanoma, Bronchoalveolar carcinoma, Pulmonary Flanagan et al. (2004), Yamano et al. (2006), Yang et al.
Exodus-2 adenocarcinomas. (2004), Yang et al. (2006)
Ckβ9
TCA-4
CCL22 MDC CCR4 Melanoma, Lung carcinoma, Ovarian carcinoma. Okada et al. (2006), Guo et al. (2002), Gao et al. (2004)
DC/β-CK
CCL27 CTACK CCR10 Melanoma, HIV-1, Influenza, HCV, HBV. Okada et al. (2006), Tyagi and Kashanchi (2012), Mohan
ILC et al. (2016)
PESKY
CCL28 MEC CCR3 HIV-1, Influenza, HCV, HBV. Tyagi and Kashanchi (2012), Mohan et al. (2016)
CCR10

Hoai et al., 2012). CCL21 is constitutively expressed in various lym- interact with the chemokine receptor CCR7, which is required for DC
phoid tissues and binds to chemokine receptor CCR7 on mature DC and and T cell trafficking into lymphoid T zones (Yasuda et al., 2007).
distinct T and B cell subpopulations. In vivo, CCL21 regulates the en- CCL21 is also made in high endothelial venules (HEVs), the pathway by
counters between dendritic and T cells and thus is a key regulator of which most lymphocytes enter the lymph nodes and Peyer’s patches. B
adaptive immune responses. CCL5 primarily controls T cell migration cells also express CCR7, and therefore CCL21 plays an important role in
toward inflammation and tissue damage, as well as Th1 differentiation. trafficking B cells into HEVs, which then progress further into lymph
Mice given tumor lysate followed with CCL5-expresseing vaccinia virus nodes and Peyer’s patches (Okada et al., 2002).
showed significant reductions in tumor growth and increased survival The CXCL12 and its corresponding receptor CXCR4 are involved in
rates. Foundational research into chemokines and cancers have re- ASCs migration towards both the bone marrow and secondary lym-
vealed key roles chemokines play in cancer immunity. The examples of phoid organs. Recent studies have shown that ASCs may migrate out of
research in this field above show how this knowledge is being applied the spleen or lymph nodes due to a loss in their ability to respond to the
to improve immune-cancer therapies. Table 1 displays the role of che- CCL19, CCL21, and CXCL13. These cells reach the boundaries of lym-
mokines as immunostimulators with respect to various infections and phoid compartments, and are then exposed to increased levels of
tumors. CXCL12, which further influences their migration toward bone marrow
and other secondary lymphoid organs (Achatz-Straussberger et al.,
2008).
7. Possible mechanisms behind the immunomodulation
properties of chemokines
7.2. Chemokines can alter APC and effector cell functions
7.1. Homing of B and T cells
Chemokines and their respective receptors are responsible for or-
It has been recently discovered that certain chemokines are con- ganizing the trafficking pattern between immunocompetent cells and
tinuously expressed in lymphoid organs, and that these chemokines can secondary lymph organs. The creation of a successful antibody immune
attract both activated and naïve lymphocytes. This subset of chemo- response is dependent on this trafficking. DCs are primarily responsible
kines with their corresponding receptors appear to play critical roles in for either the creation or prevention of immune responses by priming or
the migration and trafficking of lymphocytes within these lymphoid inducing tolerance in T cells (Coscia and Biragyn, 2004).
organs (Okada et al., 2006; Miyasaka and Tanaka, 2004; von Andrian Chemokines have been shown to play a major role in the recruit-
and Mempel, 2003). ment of DCs to desired locations. The iDCs (immature) can be char-
Both CCL19 and CCL21 are produced by T zone stromal cells and acterized by high levels of both phagocytic ability and MHC molecules,

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but are lacking in co-stimulatory molecules (McColl, 2002). Various was also used as an adjuvant in some of the studies and showed a
chemokines such as CCL21, CCL7, and CCL20 cause the migration of significant increase in the antigen-specific immunity in animals. During
iDCs to sites of inflammation. Once iDCs arrive to inflamed or damaged HSV infection, researchers found that CCL2, CCL3 and CCL4 provide
tissue sites, they acquire an antigen, upregulate co-stimulatory mole- valuable adjuvanticity in mucosal DNA vaccination by providing
cules and can activate into mDCs (mature) (Dieu et al., 1998). These linkage signals between innate and adaptive immunity. CCL3 as an
iDCs express CCR1, CCR2, CCR5, and CCR6, which are responsible for adjuvant in the form of an expression plasmid was concluded to be a
their chemotactic migration. The mDCs downregulate these same re- promising target in developing HSV-2 vaccine (Harandi et al., 2001).
ceptors while upregulating CCR7, CCL19 and CCL21 to guide the an- CCL21 has been used as a plasmid DNA adjuvant to enhance Tyrosinase
tigen loaded mDCs to the secondary lymph organs for further proces- related protein 2 (TRP2)-specific immunity in a melanoma cancer
sing (Sallusto et al., 1999). The formation of antigen-specific killer cells vaccine (Yamano et al., 2006).
requires cytotoxic T cells to be primed by DCs and then licensed by Another advantage of chemokines versus traditional adjuvants is
CD4+ T cells. As such, naïve CD8+ and CD4+ T cells, are constantly their malleability. Chemokines are proteins that can be easily modified
looking for DCs presenting a matching antigen (Mandl et al., 2012). to add new functionality and form fusion proteins. CXCL10 has been
After receiving an inflammatory or danger signal, DCs begin to mature, studied as a GPI-anchored fusion protein in an NK based tumor therapy.
and enhance the CCR7 expression (Dieu et al., 1998; Chan et al., 1999; CXCL10 is secreted by leukocytes and tissue cells and functions as a
Sallusto et al., 1998). Studies showed that a deficiency of CCR7 or its chemoattractant, mainly for lymphocytes and evokes a range of in-
ligands, CCL19 and CCL21, leads to impaired DC migration (Forster flammatory responses. The results suggest enhanced recruitment of NK
et al., 1999; Gunn et al., 1999). In addition, a recent study indicates that cells by CXCL10-mucin-GPI. This class of fusion proteins represents a
steady-state trafficking of skin DCs to the draining lymph nodes in novel adjuvant in cellular immunotherapy (Muenchmeier et al., 2013).
peripheral tissues is also regulated by CCR7-mediated signaling and In another study, the CXCL10-mucin-GPI chimera fusion protein
demonstrated that CCR7 plays a key role in governing the trafficking of showed promising results as a future cellular immunotherapy (Nelson
DCs under both inflammatory and steady-state conditions (Ohl et al., and Muenchmeier, 2013). The underlying concept of a chemokine head
2004). fused to the mucin domain and a GPI anchor signal sequence may be
Several chemokines are essential to this behavior. CCL3 and CCL4 expanded into a broader family of reagents that will allow targeted
are secreted by DCs in inflamed lymph nodes, which guide CCR5 ex- recruitment of cells in various settings.
pressing naïve CD8+ T cells to locations where they can interact with Chemokine CXCL10 and CCL7 fused to lymphoma Ig variable re-
APCs (Hugues et al., 2007). Simultaneously, mDCs are secreting CCL19 gions (sFv) elicited in vitro chemotactic responses and in vivo in-
which increases the antigenic response and scanning action of naïve flammatory responses. Immunity was not elicited by controls, including
CD4+ T cells (Kaiser et al., 2005). By increasing CD8+ T cells’ inter- fusions with irrelevant sFv; fusions with a truncated chemokine that
action with APCs and increasing CD4+ T cells scanning action, the lacked receptor binding and chemotactic activity, mixtures of free
result is effective maturation of cytotoxic T cells. Once the T cells have chemokine and sFv proteins, or naked DNA plasmid vaccines encoding
differentiated, the trafficking pattern needs to return these cells back to unlinked sFv and chemokine. Researchers concluded in their study that
the target area. This is accomplished by the downregulation of CCR7 sFv-chemokine fusion proteins induce protective and strong T cell de-
and the upregulation of CCR1, CCR2, CCR3, CCR5, and CXCR3, which pendent antitumor immunity (Biragyn et al., 1999).
are receptors unique to chemokines expressed by cells in the target area Simultaneously, the soluble form of CX3CL1 has also been studied,
(Kaiser et al., 2005). which helps to recruit lymphocytes, DCs, and NK cells. In this meta-
Through this interplay of constitutively expressed chemokines in static colon cancer mouse model study, CX3CL1 exerts strong antitumor
tissues and the up and down regulation of cell surface receptors, im- activity in its soluble form too when compared with other vaccine
mune cells can migrate to sites of inflammation, acquire an antigen, formulations. CX3CL1 as a soluble protein causes significant increase in
mature, and then migrate again back into target tissues. The sources of the migration of NK cells, cytotoxic T lymphocytes, and macrophages
chemokines act as beacons, guiding cells from a distance to an im- (Robinson et al., 2003).
portant local area where the environment is conducive to the devel- In our recent finding, we studied a GPI-anchored form of CCL28 in
opment of healthy immune responses. We have discussed in detail the VLPs and compared the results with its soluble form in mice. We ob-
functions of various chemokines in context with different diseases in- served that the GPI-anchored form provides effective and long-lasting
cluding cancers in Table 2. mucosal immunity against H3N2 viruses when compared to the soluble
form of CCL28 (Mohan et al., 2016; Mohan et al., 2017).
8. Current advancement in using chemokines as adjuvants The intrinsic attributes of chemokines allow for them to be used as
adjuvants in different forms, whether that be a soluble form coadmi-
Chemokines display their immunomodulatory functions with many nistered with the vaccine, a plasmid DNA adjuvant, or a chimeric re-
pathogens. In the investigation of these topics, researchers were focused combinant protein. This makes the use of chemokines as adjuvants in-
on the various aspects of chemokines in different forms, exhibiting credibly adaptable to any vaccine application.
different functions, and applications through various routes of vacci-
nations. 8.2. Chemokines adjuvants delivered through different vaccination routes

8.1. Chemokine as an adjuvant in different forms The route of vaccination is important in influencing immune re-
sponses at the initial site of pathogen invasion where protection is most
DNA-based vaccines induce antigen-specific cellular and humoral effective. Immune responses required for protection can differ vastly
immune responses by encoding a foreign antigen. Despite many ad- depending on the individual pathogen, adjuvant used or route of vac-
vantages, the low-immunogenicity and poor translation from rodent to cinations (Belyakov and Ahlers, 2009).
human trials have revealed a vital necessity for adjuvants. For the generation of effective protective immunity, scientists have
In many findings, chemokine expression plasmids or DNA based used various routes of vaccination when chemokines were used as im-
chemokine vaccine formulations showed substantial increases in the munostimulating agents. For instance, CCL2, CCL3, CCL4, CXCL2,
antigen-specific immunity. For example, CCL27 and CCL28 were used CXCL3 and many more chemokines were studied as adjuvants and de-
as adjuvants in the form of an expression plasmid with HIV-1 DNA livered in animals through the intranasal route, which is one of the best
vaccines and significant enhancement in the protective immune re- vaccination routes for the development of mucosal immunity (Belyakov
sponses were observed (Rainone et al., 2011). CCL5 expressing plasmid and Ahlers, 2009).

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Table 2
Different functions of Chemokines.

Chemokines Functions References

CCL1 CCL1 attracts monocytes, NK cells, immature B cells, and immature dendritic cells by interacting with a Roos et al. (1997)
cell surface chemokine receptor called CCR8.
CCL2 CCL2 influences macrophage, monocyte, NK cell, basophil and T lymphocyte infiltration, which express Kuroda et al. (2005)
chemokine receptors CCR2.
CCL3 CCL3 functions as a chemoattractant for inflammatory cells and modulates functions of monocytes, B and T Guan et al. (2001), Wolpe et al. (1988)
lymphocytes, and also influences hematopoietic stem/progenitor cell growth.
CCL4 CCL4 is a chemoattractant for natural killer cells, monocytes and a variety of other immune cells. CCL4 is a Cocchi et al. (1995)
major HIV-suppressive factor produced by CD8+ T cells.
CCL5 CCL5 promotes the migration and activation of several types of leukocytes expressing CCR1, CCR3, and Lapteva and Huang (2010)
CCR5. CCL5 has a strong chemotactic activity towards multiple immune cells, such as eosinophils,
basophils, mast cells, monocytes, CTLs, naive CD4+ T cells, stimulated CD4+ and CD8+ T cells, NK cells,
memory CD45RO+ T cells and immature DCs.
CCL7 CCL7 can chemoattract a large panel of leukocytes including monocytes, eosinophils, basophils, DCs, NK Wetzel et al. (2007), Fioretti et al. (1998)
cells and activated T lymphocytes by interaction with the CCR1, CCR2 and CCR3 receptors.
CCL16 CCL16 chemoattracts monocytes, lymphocytes and PMNs by binding to CCR1 and CCR8 receptors. Li et al. (2003)
CCL17 This chemokine specifically binds and induces chemotaxis in T cells and elicits its effects by interacting
with the chemokine receptor CCR4.
CCL19 CCL19, produced by a subset of DCs and possibly by other nonlymphoid cells in T cell areas of lymphoid Braun et al. (2000)
tissues, strongly attracts naive T cells and DCs and plays a central role in regulating the encounters
between DC and T cells in secondary lymphoid tissues via interaction with CCR7.
CCL20 CCL20 elicits its effects on its target cells by binding and activating the chemokine receptor CCR6 that is Baba et al. (1997), Guo et al. (2001)
expressed on most B cells, effector/memory T cells and some subsets of DCs.
CCL21 The CCL21 interaction with CCR7 leads to establishment of the functional secondary lymphoid tissue Liang et al. (2007)
microenvironment and angiogenesis blockage, respectively.
CCL22 CCL22 is an active chemokine on DCs, NK cells and Th2 lymphocytes. This chemokine is secreted Vulcano et al. (2001)
predominantly by DCs, and exerts its effects by interacting with CCR4.
CCL27 CCL27 is expressed in the skin and selectively chemoattracts cutaneous lymphocyte-associated Ag (CLA)+ Morales et al. (1999)
memory T and Langerhans cells. It can regulate migration of skin-homing lymphocytes.
CCL28 CCL28 plays a role in the physiology of extracutaneous epithelial tissues, including diverse mucosal Hieshima et al. (2003), John et al. (2005), Kunkel
organs. CCL28 has dual roles in mucosal immunity as a chemoattractant for cells expressing CCR10 and/or et al. (2003), Rodriguez et al. (2004)
CCR3 such as plasma cells and also as a broad-spectrum antimicrobial protein secreted into low-salt body
fluids. It drives the mucosal homing of T and B lymphocytes that express CCR10, and the migration of
eosinophils expressing CCR3.

The delivery of vaccine components through other routes such as phenomena (Perricone et al., 2013). The recent cases of narcolepsy
intradermal, subcutaneous, and intramuscular are also common routes observed during the 2009 pandemic influenza campaign with an AS03-
of immunization. In a recent study, CXCL9 and CXCL10 showed sig- adjuvanted vaccine have further heightened awareness.
nificantly higher adjuvanticity when immunized intratumorally Certain adjuvants have specific receptor targets that strongly sti-
(Bronger et al., 2016). As CCL28 and CCL27 showed immunoprotective mulate the immune system via pattern recognition receptors (PRRs),
effects on mucosal sites and epidermal sites, their applications through including toll-like receptors (TLRs), and stimulating such targets could
various mucosal and skin routes have been studied (Rainone et al., theoretically increase the risk of initiation or progression of systemic
2011). In addition, successful vaccination approaches using epicuta- AID (Marshak-Rothstein, 2006). Chemokines are produced naturally by
neous and microneedle delivery platforms have been thoroughly stu- the body’s own immune system, could serve as potentially safer and
died in models of influenza with some of the chemokines. more reliable adjuvant options versus synthetic adjuvants. The immune
Again, chemokines make very flexible adjuvants because they can “hyperactive” should not be a potential drawback when chemokines, in
be delivered by any possible vaccination route. In some cases, a che- an optimum dose, are used as adjuvant. Chemokines induced antigen-
mokine may be particularly well suited to one route or another de- specific immune responses by two possible mechanism of actions: 1) by
pending on the immune cells it interacts with. Matching chemokines to recruiting various immune cells at the site of injection, or/and 2)
their optimal vaccination route provides another area of possibilities in modulating effector functions of these chemoattracted immune cells.
chemokine adjuvant research. These two mechanisms should not generate toxicity. Though the gen-
eration of toxicity with some of vaccine studies containing chemokines
9. Chemokines and toxicity have been highlighted, this could be related with the high dose of
chemokines used in the vaccines’ formulations and more work should
Vaccines play an important role in modern medicine in the pre- be done to identify optimal dosages of chemokine adjuvants.
vention of diseases including cancers. Most of the available vaccines
work under the basic premise that the immune system becomes primed 10. Therapeutic potential of chemokines
against a possible future exposure upon vaccination, therefore, pro-
viding protection to an individual. Developing adjuvants is challenging, A major role of chemokines is to mediate various immune cell mi-
and adjuvants are under regulatory scrutiny because of theoretical and gration through interaction with G-protein-coupled receptors. As a
reported safety concerns (Sesardic and Dobbelaer, 2004). potential therapeutic, various delivery systems have been developed to
As mention earlier in the review, the addition of synthetic adjuvants utilize the chemokine properties for combating disease. Chemokines
may induce “hyperactive” immune responses and can increase the risk have been administered through various delivery strategies such as
of developing autoimmune diseases (AID). Possible safety concerns viral and mutant viral vectors expressing chemokines, genetically
have arisen from studies in which adjuvants have induced AID in var- modified dendritic cells with chemokine or chemokine receptors, en-
ious animal models and from reports that diverse compounds with gineered chemokine-expressing tumor cells and pDNA encoding che-
adjuvant activity could be associated with silicosis, Gulf war syndrome mokines.
(GWS), macrophagic myofasciitis (MMF), and post-vaccination In addition, chemokines induce more effective antitumor immunity

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when used as adjuvants. In this regard, chemokines are codelivered 13. Conclusion
along with antigens or fused as a targeting unit with antigenic moieties.
In the current review, we have discussed in detail the im- The major purpose of this review is to introduce chemokines’ ap-
munomodulatory properties of various chemokines with respect to plication in preventing or curing various infectious diseases and can-
many diseases and cancers. The actions of chemokines may be in- cers. Many delivery systems including plasmid DNA encoding chemo-
trinsically therapeutic or immunomodulatory. For example, it has been kines, chemokine targeting of DCs or tumor cells, and chemokine
shown that epigenetic repression of CXCL9 and CXCL10 is a tumor expressing live vaccines have been evaluated in various experimental
immune-evasion mechanism. However, removing this repression or models. Currently, many studies are showing significant benefits from
otherwise creating high expression of these two chemokines was found the use of a combination of various chemokines in combating cancers or
to increase T cell tumor infiltration and slowed down tumor progression infectious diseases. Some studies applied conventional therapies such as
(Peng et al., 2015). Simultaneously, a research group found that a single cytotoxic chemotherapy together with chemokine-based im-
injection of COAM into a tumor site demonstrates potent anti-tumor munotherapy. The principal aims of combination therapies are to sy-
properties that were mediated through chemokine induction. COAM nergistically enhance antibody responses and potentially reduce toxic
both binds to CXCL6 and upregulates the expression of CXCL6. This side-effects.
induction and concentration of CXCL6 subsequently leads to a massive Chemokine based adjuvants have a great deal of potential. These
chemotactic recruitment of anti-tumor myeloid cells into the tumor adjuvants can be fine-tuned with genetic manipulation to produce the
(Piccard et al., 2012). ideal chimeric chemokine for the application at hand. The versatility of
While the beneficial actions of chemokines revolve around their delivery routes means chemokine adjuvants have to potential to be used
regulation of immune cell migration, the results of the immune cell in a broad range of applications. Ultimately, the chemotactic signaling
migration may be thought of as either therapeutic or of adjuvant of chemokines combined with their flexibility in form and delivery has
properties depending on the application. allowed researchers to begin developing vaccines and cures that would
not have been possible before. This potency is evident in the wide
spectrum of applications presented within this review: HIV, influenza,
11. Clinical trials with chemokines as adjuvants HSV, carcinomas, melanomas, and blastomas. As research progresses
and data accumulates, chemokines will become more powerful and
The best hope for the prevention of infectious diseases, especially in prevalent tools in immunology, undoubtable usurping the old guard of
resource-poor countries, is an effective preventive vaccine. Although aluminum based adjuvants in the process.
generalized adjuvants have long been used to enhance the immune
response to vaccines, the use of chemokine adjuvants with specific Conflicts of interests
immune modulatory functions allows for the manipulation of the host
response to both enhance overall immunogenicity and direct the nature There are no known conflicts of interests.
of the response toward a Th1 or Th2 pathway.
Chemokine formulations with various immunogens are being con- Acknowledgement
sidered for in vitro studies, pre-clinical and clinical trials. For example, a
genetically modified leukemia/lymphoma vaccine that expresses CCL3 We thank Gilbert X. Gonzalez for his help in reviewing of the
plus IL-2 or CCL3 plus GM-CSF has been studied (Zibert et al., 2004). manuscript.
Intradermal injection of adenovirus-CCL21 transduced class I peptide-
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